Detection of clade 2.3.4.4 highly pathogenic avian influenza H5 viruses in healthy wild birds in the Hadeji‐Nguru wetland, Nigeria 2022

Abstract Background The introduction of multiple avian influenza virus (AIV) subtypes into Nigeria has resulted in several poultry outbreaks purportedly linked to trade and wild birds. The role of wild birds in perpetuating AIV in Nigeria was, therefore, elucidated. Methods A cross‐sectional study was conducted among wild aquatic bird species at the Hadejia‐Nguru wetlands in Northeastern Nigeria between March and April 2022. A total of 452 swabs (226 cloacae and 226 oropharyngeal) were collected using a mist net to capture the birds. These samples were tested by RT‐qPCR, followed by sequencing. Results Highly pathogenic AIV of the H5N1 subtype was identified in clinically healthy wild bird species, namely, African jacana, ruff, spur‐winged goose, squared‐tailed nightjar, white‐faced whistling ducks, and white stork. A prevalence of 11.1% (25/226) was recorded. Phylogenetic analysis of the complete HA gene segment indicated the presence of clade 2.3.4.4b. However, these H5N1 viruses characterized from these wild birds cluster separately from the H5N1 viruses characterized in Nigerian poultry since early 2021. Specifically, the viruses form two distinct genetic groups both linked with the Eurasian H5N1 gene pool but likely resulting from two distinct introductions of the virus in the region. Whole‐genome characterization of the viruses reveals the presence of mammalian adaptive marker E627K in two Afro‐tropical resident aquatic ducks. This has zoonotic potential. Conclusion Our findings highlight the key role of surveillance in wild birds to monitor the diversity of viruses in this area, provide the foundations of epidemiological understanding, and facilitate risk assessment.


| INTRODUCTION
Wetlands are terrestrial or semi-terrestrial ecosystems characterized by low drainage, slow waters, or seldom standing water bodies filled with soil.Wetlands are very important and valuable components of the ecosystem.They serve as a habitat for man and animals, a source of food, shelter, and other ecosystem functions.For many faunas, wetlands are the life-enhancing systems of the environment; they consist of direct and indirect components.3][4] In Nigeria, several coastal and inland wetlands serve as congregation sites for both migratory and resident waterfowl.
The Hadejia-Nguru wetland is the largest in Northeastern Nigeria with an estimated coverage area of 3500 km 2 . 5These wetlands serve as seasonal shelters and migratory routes for approximately 5.4 million wild aquatic birds, making seasonal movements between the temperate zone and the tropics during winter. 6Over 80 wild bird species have been caught in this location during bird counts and avian influenza surveillance. 7The interaction between the migratory birds and the indigenous resident birds during stop-overs at the wetlands provides a veritable platform for the exchange of influenza A viruses and their genetic materials.
][10][11][12][13] The Hadejia-Nguru wetlands are located about 220 km from the first HPAI H5N1 outbreaks reported in Kano State in January 2015 and could be a source of infection and maintenance of the virus. 2,8,14Although there is low transmissibility of HPAIV from birds to humans and high fatality has been reported in humans in previous epidemics, the potential for a mutant strain of HPAIV to become pandemic through spillover from infected migratory birds poses a threat to public health. 15veral phylogenetically distinct sub-lineages of the HPAIV have been reported in wild migratory birds. 16In Nigeria, the HPAIV H5N1 belonging to the genetic clade 2.3.2.1c was reported in 2015 from samples collected from backyard poultry as well as live bird market (LBM). 17,18Before that, however, outbreaks of HPAIV subtype H5N1 with genetic clades 2.2 and 2.2.1 had occurred in the country in 2006-2008. 19,20These outbreaks have had a devastating impact on the poultry industry as well as international trade in poultry and poultry products. 21Owing to the antigenic variability and the segmented nature of the influenza virus genome, several reassortant strains and subtypes of the AIVs have been reported in Nigerian poultry. 22These include the H5N1, [17][18][19]21 H5N2, 23 H5N8, 12,24 H5N6, 25 and H9N2. 26Previously, Gaidet et al. 27 had reported the detection of the H5N2 subtype of the AIV with highly pathogenic characteristics in apparently healthy wild waterfowl species sampled in the Hadejia-Nguru wetlands in Nigeria.It is speculated that AIV is not yet endemic in Nigeria, but rather that there are repeated introductions by migratory wild birds (Meseko pers. com). FoFor over a decade, no active AIV surveillance has been implemented in the wild bird population hosted by one of the most relevant wetlands of the Western African region, Hadejia-Nguru. Ths has negatively impacted the ability to design strategies to recognize and mitigate potential AI threats for the region coming from wildlife populations.Therefore, the objective of the surveillance effort described herein was to identify the circulating AIV subtypes in the wild birds in the Hadejia-Nguru wetlands and to gain deeper insight into the ecology of the virus. Mag use of the unique collection of samples from this important wild bird area, we provide new insights into the epidemiology of the disease in wild migratory and resident birds in Northeastern Nigeria.

| Description of study location
The Hadejia-Nguru wetlands are a wide expanse of floodplain wetlands situated in northeast Nigeria, in the Sudan-Sahelian region, which is the zone between the Sudanian Savanna in the south and the Sahel in the North.It is one of Nigeria's major wetlands, covering an area of about 3500 km 2 with an altitude of 152-305 m above sea level and a depth of about 1.6-1.7 m.The annual rainfall ranges between 200 and 600 mm, from late May to September.The mean minimum temperature ranges from 12 C in December to January, reaching a maximum of 40 C in April. 5,28These wetlands are drained by two major rivers, the Hadejia and the Jama'are, which flow and converge into Lake Chad (Figure 1).The wetlands enclose Kano, Jigawa, Yobe, Gombe, and Bauchi States.It is an ecologically and economically rich habitat for the biodiversity of various fauna and flora.
The area is a major tourist location for the Palaearctic and Afrotropical migrant water birds.Large numbers of diverse migratory species mix with resident wild birds and domestic waterfowl. 7

| Study design, sample collection, and processing
A cross-sectional study design was adopted for this survey.Wild birds were sampled opportunistically using a mist net and bird-call sound playbacks.Birds that get entangled are quickly removed to avoid injury, and a swab sample is collected.The birds are released back into their natural habitat as a key conservation element.All the birds sampled were apparently healthy (Figure 2A-C).
Bird species were identified with the aid of bird atlas.Tracheal and cloacae swabs were collected from migratory and resident aquatic wild birds between March and April 2022.These samples were collected into 1-mL glycerol-based virus transport medium and transported on ice to the National Veterinary Research Institute, Vom Regional Support Laboratory for Animal Influenza and Newcastle Disease, where the samples were analyzed using a standardized protocol as previously described. 25,29

| Molecular detection of avian influenza virus
Using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), total RNA extraction was conducted based on the manufacturer's instructions.To detect the influenza A virus matrix gene (M-gene), 30 the RNA extracts were tested by the quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) using the QuanTitect ® Multiplex RT-PCR kit (Qiagen ® , Hilden Germany) in the Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany).The M-gene-positive samples were subsequently sub-typed for the hemagglutinin gene (HA) using an established H5N1 duplex protocol. 31The QuantiTect ® Multiplex RT-PCR kit was used (Qiagen, Hilden Germany).Briefly, the master mix reagents included a 5Â PCR buffer, H5 probe mix (10 μm), N1 probe mix (10 μm), dNTP (10 μm), Mgcl2 (25 μm), Rnasin (40 μm), and enzyme mix (1 μm).The RNA template (5 μm) was added to the mix and was amplified using the following cycling conditions: 50 C for 30 min, 95 C for 15 min, 95 C for 30 s, 56 C for 30 s, and 72 C for 10 s with 45 cycles/repeats.The fluorescence for H5 was acquired at the HEX, whereas the N1 was at the FAM.These represent the targeted probes/dye.The Ct value less than or equal to 35 for each sample was considered positive for H5 and N1 genes respectively.

| Virus isolation
All samples that were positive for M-gene were selected for virus isolation according to a standard method. 29The samples were inoculated into 9-to 11-day chicken embryonated eggs obtained from a specific pathogen-free (SPF) flock.The inoculated eggs were incubated at 37 C and candled at 24-h intervals for 5 days.Eggs with dead embryos within the first 24 h post-inoculation were discarded as nonspecific mortalities.Subsequently, eggs with dead embryos were set aside in a refrigerator at +4 C. The resultant allantoic fluid containing the virus was harvested and tested for the presence of hemagglutinating activities serologically. 29

| Genome sequencing and phylogenetic analysis
Seventeen HPAI H5 positive clinical specimens were sent to the European, WOAH, and FAO reference laboratory for avian influenza (AI) in Italy (Istituto Zooprofilattico Sperimentale delle Venezie) for sequencing and genetic data analysis.A target RT-PCR approach was used to amplify influenza A virus whole genomes as previously described. 12Sequencing libraries were obtained using the Illumina DNA prep kit (Illumina, San Diego, CA, USA) and sequenced on MiSeq instrument using a 2 Â 300 bp PE mode.Raw sequencing reads produced by MiSeq instrument were cleaned with Trimmomatic v0.32 with minimum quality 20.Illumina DNA prep adapter sequences were clipped from reads using scythe v0.991 (https://github.com/vsbuffalo/scythe), and amplification primers were removed using sickle v1.33 (https://github.com/najoshi/sickle). Reads shorter than 80 bp or unpaired were discarded.The cleaned reads were aligned against a reference genome using the MEM algorithm from BWA v0.7.12-r1039.Picard tools v2.1.0(http://broadinstitute.github.io/picard/) and GATK v3.530-32 32 were used to improve alignment quality, correct potential errors, and recalibrate base quality score.LoFreq v2.1.2.33 33 was used to call single nucleotide polymorphisms that were reported in a vcf (Variant Call Format) file.The generated vcf file was then used to produce the consensus sequences using an in-house script.Briefly, this script calls the base for each position with a coverage >10Â, considering all the polymorphisms with a frequency higher than 25%."N" is assigned to all positions with a coverage lower than 10 reads.

| RESULTS
In all, a total of 452 swab samples from 226 birds were collected (226 tracheal and 226 cloaca swabs) from 30 different bird species.
T A B L E 1 Distribution of bird species, migrant type, molecular analysis results, and the prevalence of AIV at Hadejia-Nguru wetlands, Northeast Nigeria.Virus isolation in embryonating SPF chicken eggs was attempted for all M gene-positive samples according to standard procedures. 29A total of 25 virus isolates were obtained and stored appropriately.

| PHYLOGENETIC AND GENOMIC ANALYSIS
Whole-genome sequences were obtained for 17 H5N1 viruses identified in wild birds in Nigerian wetlands.Analysis of the hemagglutinin (HA) gene segment confirms the presence of a cleavage site motif typical of the HPAI viruses (KRRKR*GLF) and shows that the HPAI H5N1 viruses from wild birds (Figure 3) belong to clade 2.3.4.4b but cluster separately from the H5N1 viruses, which have been circulating in the Nigerian poultry population since early 2021. 36Specifically, the viruses identified in wild birds form two distinct groups in all the phylogenetic trees provisionally named here A and B groups (Figure 2 and Figures S1-S7).Most of the viruses detected in wild birds in the Hadejia-Nguru wetlands cluster together in group B with viruses identified in wild and domestic birds in Senegal in December 2020-   3).The mutations identified in the HA protein, which have proved to increase in vitro binding to humantype receptors (i.e., S133A, S154N, T156A, S107R, T108I, K218Q, and S223R) and the NS1 changes, described to increase replication in mammalian cells, have been identified in the majority of the A(H5N1) viruses circulating in Europe since October 2020; same applies to the mutations identified in the PB1 and PA genes, which have been already reported in 2.3.4.4b viruses detected in Europe. 37It is, therefore, likely that these mutations have been acquired elsewhere before being introduced in Nigerian wildlife.

| DISCUSSION
In this study, 30 different wild bird species were identified; these include, among others, afro-tropical ducks such as white-faced whistling ducks, spur-winged geese, and fulvous whistling ducks.Also found were waders such as ruff, common snip, herons such as greyhead heron, common squacco heron, and black-headed heron T A B L E 3 Amino acid markers associated with specific phenotypic effects found in the genome of HPAI H5N1 viruses sequenced in the current study.1).This finding corroborates previous reports of the species of wild birds found in the Hadejia-Nguru wetland. 7,11,38African jacana (Actophilornis africanus), ruff (Calidris pugnax), spur-winged goose (Plectropterus gambensis), squared-tailed nightjar (Caprimulgus fossii), white stork (Ciconia Ciconia), and white-faced whistling ducks (WFWD) (Dendrocygna viduata) are Afrotropical and Palearctic wild bird species found to harbor HPAI of the H5N1 subtype in the study.
Spur-winged goose and the WFWD (Anatidae species) were previously reported to harbor the low pathogenic AIVs. 39,40These ducks exhibit similar foraging behavior and share the same feeding and roosting habitat.Their gregarious nature and foraging in shallow waters expose them to AIV infection.The detection of HPAI from these species within the region partly agrees with the report of Gaidet et al., 27 who earlier detected the H5N2 subtype of the AIV with a highly pathogenic characteristic in apparently healthy wild waterfowl from the Hadejia-Nguru wetland.These H5N2 viruses were genetically related to the H5 low pathogenic strains found in Eurasian wild and domestic ducks.In our study, H5N1 virus belonging to clade 2.3.4.4b was detected over a decade later in the same location and presented even in this case the highest similarities with Eurasian viruses confirming these wetlands as an area at risk of introduction of viruses from Eurasia.
The African jacana species, from which AIV of the H5N1 subtype was also detected, are waders common in freshwater wetlands in sub-Saharan Africa.These intra-African migrants walk on floating vegetation to feed on aquatic insects.The detection of AIV in this species of wild birds disagrees with the studies by Poen et al. 41 who reported that no AIV was detected in the African jacana species screened in South Africa.HPAI was also detected in the cloaca and oropharyngeal samples from white stork species (Ciconia Ciconia).reported also in Europe suggesting that white stork could spread the virus along its migratory route. 37ff (Calidris pugnax) was another migratory bird species found infected by H5N1 in this study.This is a medium-sized shorebird that breeds mostly across northern Europe and winter in Africa.These gregarious bird species travel over 14,000 km annually in large flocks from Europe to the wintering ground in West Africa with few stopovers. 43The detection of HPAI H5N1 in this healthy bird indicates that birds belonging to this species may have been also involved in the dissemination of the virus from its breeding site in Europe.
Through phylogenetic analysis, we were able to demonstrate the existence of a genetic relationship between the viruses identified in our study and the ones previously detected in the Eurasian territories.
In particular, we identified the introduction of at least two distinct genetic clusters of the H5N1 virus (groups A and B) in the wild bird population of Hadejia-Nguru wetlands.Specifically, 12 viruses isolated from five different wild bird species cluster together (group B) and with viruses identified in wild and domestic birds in Europe (2020-2021) and in Western (2020-2022) and Southern African regions (2021-2022). 44The chronological order of the events and the phylog- ). 37,45 This observation suggests that these mutations with potential public health implications likely emerge upon transmission to mammals.So far, no 2.3.4.4bH5N1 infected mammals have been reported in West Africa, but these findings highlight the risk that undetected spillover events may have been occurring also in this region.Furthermore, limited surveillance of AIV in mammals in Nigeria may cause missed opportunities for early detection and early warning of emerging novel influenza viruses with this or another type of important mutation.In a previous report on AIV in mammals in Nigeria, 11 the exposure of domestic pigs to the H5N1 virus was described, which highlights the potential public health and pandemic risk by unreported but circulating AIV with mammalian adaptations.
Therefore, the need for expansion and enhancement of surveillance in poultry and mammals cannot be over-emphasized due to interspecies transmission and the occurrence of mutations of note.
Although no mortality events were observed in the wild birds sampled in the current study, the virus was actively replicating in the respiratory and digestive tract as evidenced in the positive results from oropharyngeal and cloaca swabs respectively.The detection of HPAI virus subtype H5N1 of clade 2.3.4.4b in apparently healthy wild birds in Nigeria confirms previous reports even in Nigeria 46 when two isolates of HPAI were recovered from the tracheal swab samples from apparently healthy ducks.There are other reports from Russia, 47 China, 48 South Korea, 49 and Italy. 50However, it is worth noting that, since early 2023, multiple mass mortality events in wild birds have been reported in other parts of West Africa including the Gambia, Senegal, and Guinea Bissau. 44,51Therefore, considering the dynamic situation of HPAI, it is pertinent to consider the conservation risk for wildlife inhabiting the Nigerian wetlands and the possibility of crosssectoral workings to include the Ministry of Environment in a onehealth approach strategy to develop and implement risk mitigation plans to protect the region.
The maintenance of this virus in the wild bird population could partly explain the sustained outbreaks of the HPAI H5N1 subtype in poultry across the country.These wild birds are sometimes captured by hunters, kept within the village, and taken to the LBM for sale.These birds eventually interact with poultry, hence the recurring outbreaks.This assertion is partly supported by reports from these authors 11,52,53 who opined that the migration of these birds enhances the geographical spread of the influenza virus and may bridge infections to resident aquatic birds and gallinaceous poultry.
It is also worthy of note that the Hadejia-Nguru wetland location is about 220 km northeast of Kano City where the re-emergence of H5N1 was previously reported in January 2015 from backyard poultry. 17ff and white storks are important Palearctic birds sampled in this study and were positive for the HPAI H5N1.This implies that as these birds migrate from one continent to another, they can transmit the virus to the resident aquatic birds, which can then further disseminate the virus across the region.In a recent report on the diversity and evolution of highly pathogenic avian influenza in Nigeria, 36  Plateau, and the upper Orashi forests in Rivers state. 54,55We, therefore, recommend sustained risk-based surveillance for AIV in wild birds for early detection, early warning alert for the poultry industry, disease mitigation, and control.

| Study limitation
This study was not able to trace the onward trajectory destinations of the migratory birds because the resources and technology of fitting satellite telemetry were limited.We recommend close monitoring of migratory and resident wild birds not only in Hadejia-Nguru but other wetlands across the country where the independent introduction of AIV has been reported in the past.A system of surveillance using local waterfowl as a sentinel to study the type, time, and frequency of introduction of AIV from wild to domestic waterfowl is recommended.

F I G U R E 1
Map of Nigeria showing wetlands, Jigawa and Yobe states, and sample collection sites.F I G U R E 2 (A) White stork, (B) spur-winged goose, and (C) ruff.
January 2021 and with viruses detected in Europe, suggesting that these viruses have been introduced concurrently into the West African region from Europe during the winter season 2020-2021.Five viruses fall within group A and cluster with H5N1 viruses, which have been circulating in Europe during the 2021-2022 epidemic wave, suggesting that it may represent a more recent introduction in Nigeria (Figure 2).All the 17 viruses characterized are descendants of the H5N1 A/Eurasian_Wigeon/Netherlands/1/2020-like genotype, which most likely emerged in Europe in 2020.Molecular analysis of the consensus sequences of the HPAI H5N1 viruses from wild birds revealed the presence of several mutations that are likely associated with an increased zoonotic potential (Table Two viruses (A/white-faced_whistling_duck/Nigeria/DA16_23VIR 8254-111/2022, A/white-faced_whistling_duck/Nigeria/DA09_23VIR 8254-104/2022) present the E627K mutation in the PB2 gene, a wellknown molecular marker of mammalian adaptation.The viruses presenting this adaptive mutation were both detected in Afro-tropical resident aquatic ducks sampled in the same location (the Dagonal waterfowl sanctuary) in April 2022 suggesting that the two affected animals may have been exposed to the same pathogen source.

F I G U R E 3
Maximum likelihood phylogenetic tree of the HA gene segment obtained in IQtree v1.6.6.The viruses analyzed in this work are marked in pink and cluster within group A (yellow box) and group B (green box).The viruses previously detected in Nigeria are shown in blue.Ultrafast bootstrap values higher than 80 are shown next to the nodes.
2023 confirm the detection of H5N1, H5N2, and H5N8 of the clade 2.3.4.4b in commercial poultry farms across different states in Nigeria.Comparison of the H5Nx viruses of the clade 2.3.4.4b viruses detected and characterized in poultry in this study 36 revealed that these poultry viruses are genetically distinct from the ones identified in Hadejia Nguru wetland.The adduced reasons for this genetic divergence can include the inadequate/limited surveillance activities for HPAI in wild birds in wetlands across the country.This has resulted in the paucity of data on the subtypes of HPAI viruses in circulation per time in these wetlands.The Hadjia-Nguru wetland though is an important internationally recognized wetland, and there are other wetlands through which wild birds can access the country to introduce the virus, which includes the Apoi Creek in Bayelsa, Forge Islands in Kebbi, Oguta Lake in Imo, Pandam and Wase lakes in Nassarawa-